Premotor networks, consisting of excitatory and inhibitory input from both sides of the spinal cord, guide the contraction of specific musculature to coordinate refined movements. In these networks, motor neurons are the sole point of output to muscle. Although one major function of spinal inhibition is to enforce left/right alternation, motor neurons also receive a substantial amount of inhibition coincident with excitation drive, of unknown functional significance. Two populations of spinal cord neurons may supply this inhibition, the genetically and anatomically defined V1 and V2b classes. The overall goal of this project is to define the activity of V1 and V2b populations in zebrafish axial circuits during locomotion and identify their participation in motor neuron spike timing modulation or another specific behavior. I propose, in Aim 1, to identify the activity of V1 and V2b neurons with concurrent in-vivo calcium imaging and electrophysiology, along with neuron ablation studies. In Aim 2, I will use electrophysiology and optogenetics to define the endogenous activity of V1 and V2b, assess their functional heterogeneity, and ultimately confirm connections to motor neurons innervating dorsal and ventral axial musculature. The proposed work utilizes state-of-the-art light sheet microscopy and optogenetics to uncover the fundamental role the V1 and V2b neurons perform in locomotion circuitry in vertebrates. These results will help understand the purpose of coincident inhibition to motor output, while determining its source and other possible functionalities of the V1 and V2b interneurons. Overall the work proposed here will expand our understanding of spinal cord premotor circuits, laying the foundation for new treatment options in injury and disease.